The Epidermal Growth Factor receptor (EGFR) is a transmembrane glycoprotein, the molecular weight of which is 170 KD. It is an expression product of oncogene C-erbB-1 (HER-1), and widely distributed on cell membrane of human tissues [Alan Wells. Molecules in focus EGF receptor. Int J Biochem Cell Biol, 1999, 31: 637-643]. It is over-expressed and (or) mutated in most tumors (For example, non-small cell lung cancer, bladder cancer, ovarian cancer, breast cancer, head and neck squamous cell carcinoma, glioblastoma, pancreatic cancer, esophageal cancer, gastric cancer, prostate cancer, etc.), and is closely related to the occurrence and development of tumor, malignant transformation, metastasis and prognosis [Jose B Why the epidermal growth factor receptor? The rational for cancer therapy [J] oncologist, 2002, 7 (4): 2-8]. Therefore, EGFR is an important target for tumor treatment. In addition, studies have shown that EGFR 287-302 epitope is only exposed in tumors expressing EGFRvIII or over-expressing EGFR, while concealed in normal tissues [Gan H K, et. Al Targeting of a conformationally exposed, tumor-specific epitope of EGFR as a strategy for cancer therapy. Cancer Res, 2012, 72 (12): 2924-2930]. It is suggested that EGFR 287-302 epitope is an ideal site for targeting EGFR-relevant tumor therapy.
Antibodies against EGFR287-302 epitope have been developed, exhibiting good tumor-specific killing effects. However, antibody therapy is limited by the in vivo half-life of antibody in the blood circulation. Generally, the half-life is not more than 23 days. Therefore, sustained administration, and/or increase in the dose is necessary for the antibody therapy of tumor, which results in the increase of cost for patients, and in some cases even results in the termination of treatment. Moreover, the therapeutic antibody, as a heterologous protein, may also be of the risk of producing allergic reactions and neutralizing anti-antibody against the therapeutic antibodies and in vivo.
More and more attention is paid to T lymphocytes in the tumor immune response. Certain effects are achieved by T lymphocyte-based adoptive immunotherapy in some tumors, and such immunotherapy can overcome the above drawbacks of antibody treatment. However, in most tumors, the efficacy is still unsatisfactory [Grupp S A, et. al. Adoptive cellular therapy. Curr Top Microbiol Immunol. 2011, 344: 149-72]. In recent years, according to the discovery that the specificity of cytotoxic T lymphocytes for recognizing target cells depends on T cell receptor (TCR), scFv of the antibody against tumor cell-associated antigen is fused with intracellular signal activation motifs, such as CD3ζ or FcεRIγ, of T cell receptor to form chimeric antigen receptor (CAR), which is genetically modified on the surface of T cell surface by certain means, such as lentivirus infection. Such CAR T cells are capable of selectively redirecting T lymphocytes to tumor cells and specifically killing tumor cells in major histocompatibility complex (MHC) and non-limiting manner. CAR T cell is a new immunotherapeutic strategy in the field of tumor immunotherapy [Schmitz M, et. al. Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer. J Biomed Biotechnol, 2010, doi:10.1155/2010/956304.]
Chimeric antigen receptor comprises extracellular binding domain, transmembrane domain and intracellular signal domain. Generally, extracellular domain comprises scFv capable of recognizing tumor-associated antigen, transmembrane domain is that of molecules, such as CD8, CD28, and immunoreceptor tyrosine-based activation motif (ITAM) such as CD3ζ (i.e., CD3 zeta, abbreviated as Z) or FcsRIy and intracellular signal domain of co-stimulating signal molecule, such as CD28, CD137, CD134 are used in intracellular signal domain.
The first generation of CAR T cells only comprises ITAM in intracellular signal domain, wherein each part of chimeric antigen receptor is connected as follows: scFv-TM-CD3ζ. This kind of CAR T cells can stimulate anti-tumor cytotoxic effects, however the secretion of cytokine is relatively law, and long-lasting anti-tumor effects can not be stimulated in the body [Zhang T. et. al. Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways, Cancer Res 2007, 67(22): 11029-11036].
In the second generation CAR T cells subsequently developed, intracellular signal domain of CD28 or CD137 (also known as 4-1BB) was added, wherein each part of chimeric antigen receptor is connected as follows: scFv-TM-CD28-ITAM or scFv-TM-/CD137-ITAM. Co-stimulating effects of B7/CD28 or 4-1BBL/CD137 occurred in intracellular signal domain lead to the sustained proliferation of T cells, and improve the level of cytokines, such as IL-2 and IFN-γ secreted by T cells, when improving the survival and anti-tumor effects of CAR T in vivo [Dotti G et. al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011, 121(5):1822-1826.].
In recent years, the third-generation of CAR T cells was developed, wherein each part of chimeric antigen receptor is connected as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, thereby further improving the survival and anti-tumor effects of CAR T in vivo [Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS, 2009, 106(9): 3360-3365].
Despite CAR T cells have an attractive prospect in tumor immunotherapy, but there are also some potential risks to be considered. For example, because some normal tissues can express specific antigens in a low level which can be recognized by CAR, CAR T cells may damage the normal tissues which express the corresponding antigen. The first case of clinically applied adoptive therapy of CAR T cells related to the antigen, carbonic anhydrase IX (CAIX) expressed on tumor cells of patients with renal cell carcinoma, which is also the first reported case about off-target effects of CAR-containing cells. After infused with CAR T cells for several times, 2-4 grade of hepatotoxicity occurs in patients. The reason is that epithelial cells of bile duct express CAIX in low level. And original clinical trial was interrupted while any evaluation on the efficacy of patients was excluded. [Stoter G et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J clin oncol, 2006, 24(13): e20-e22.; Ngo M C., et al. Ex vivo gene transfer for improved adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7]. Additionally, excessive co-stimulating signals in CAR will reduce the threshold for the activation of effector cells, so that the gene-modified T cells with a low level of antigen or without antigen triggering conditions may also be activated, thereby resulting in the release of a large number of cytokines and leading the so-called “cytokine storm”. Such signal leakage will result in off-target cytotoxicity, thereby producing non-specific damage to tissues. For example, in the clinic process of treating an advanced colorectal cancer patient with liver and lung metastases by using the third-generation of CAR targeting Her2, so-called “cytokine storm” triggered by the low expression of Her2 in normal lung tissue resulted in sudden death of the patient [Morgan R A., et al. Report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb2. Molecular Therapy, 2010, 18 (4): 843-851].
Therefore, there is a strong demand in the art to the tumor therapy using CAR T lymphocytes for overcome the above mentioned drawbacks.